Field of the Invention
The present invention relates to a process for generating hepatocyte cells derived from human induced pluripotent stem cells (iPSCs). In particular, the process can induce the differentiation of iPS cells into functional hepatocyte cells in less than 20 days, especially less than 15 days. The iPSC-derived hepatocyte cells have the similar gene expression profile to mature hepatocytes, and can be used in treatment of liver diseases.
Brief Discussion of the Related Art
Viral hepatitis or drugs often cause liver injury and cirrhosis. Liver transplantation is the only effective treatment for end-stage liver diseases; however, serious side effects of chronic immunosuppression and lack of suitable donor livers are major obstacles to liver transplantation. Reprogramming of mouse and human somatic cells to become induced pluripotent stem cells (iPSCs) has recently been achieved by viral transduction using four transcription factors (Takahashi K, et al. Cell 131:861-872, 2007). Unlike human embryonic stem (ES) cells, human iPSCs provide an alternative approach that avoids the controversies associated with the use of human embryos to obtain pluripotent ES cells. Although their gene expression pattern is not identical to human ES cells, human iPSCs are pluripotent and able to differentiate into most, if not all, cell types of the body. Therefore, human iPSC-derived somatic cells, such as hepatocytes, would be able to serve as an alternative source for liver transplantation, as well as help with toxicity screening during drug discovery.
During embryonic development, epiblast cells receive sequential developmental cues and undergo epithelial-to-mesenchymal transition to generate mesoderm or definitive endoderm. Several studies have successfully generated hepatocyte-like cells from human ES cells (Rambhatla L, et al. Cell Transplant 12:1-11, 2003; Cai J, et al. HEPATOLOGY 45:1229-1239, 2007; Hay D C, et al. Cloning Stem Cells 9:51-62, 2007; Touboul T, et al. HEPATOLOGY 51:1754-1765, 2010); and human iPSCs (Song Z, et al. Cell Res 19:1233-1242, 2009; Sullivan G J, et al. HEPATOLOGY 51:329-335, 2010; Ghodsizadeh A, et al. Stem Cell Rev 6:622-632, 2010; Si-Tayeb K, et al. HEPATOLOGY 51:297-305, 2010) in vitro.
However, most of previous studies have focused on how to develop an efficient differentiation protocol with which to generate functional hepatocyte-like cells. The conventional differentiation protocols require a period of at least 3 weeks (about 21 days or more) to generate functional hepatocyte-like cells. For the clinical usage of liver cell transplantation as an alternative option for treatment of liver diseases, it is needed to develop a rapid differentiation process to induce iPSCs into mature hepatocyte cells in less than 20 days (preferably less than 15 days).
Under the culture conditions for the generation of the hepatocyte-like cells, human ES cells or human iPSCs are first differentiated into definitive endoderm, followed by generation of mature hepatocytes that express stage- and tissue-specific genes (D'Amour K A, et al. Nat Biotechnol 23:1534-1541, 2005).
Hepatocyte growth factor (HGF) is essential for the development of liver. Previous studies demonstrated that HGF knockout mice fail to completely develop their liver architecture, with a loosened liver structure and dissociation of the parenchymal cells in the mouse model (Schmidt C, et al. Nature 373:699-702, 1995). HGF and its receptor c-MET also exert several important functions that are associated with cell proliferation, survival, motility, invasion, and morphogenesis (Liu X, Newton R C, Scherle P A. Trends Mol Med 16:37-45, 2010). In addition to its pathophysiological functions, HGF has been shown to induce scattering of epithelial cells by up-regulating expression of Snail, which is a transcription repressor that directly targets E-cadherin. However, HGF-associated molecular mechanisms during embryonic development are still poorly understood. In our previously published study, we successfully generated hepatocyte-like cells from mesenchymal stem cells in vitro by a two-step protocol involving HGF and oncostatin M (Lee K D, et al., 2004, Hepatology 40:1275-1284).